Radiopharmaceutical bacteriostats

ABSTRACT

Benzalkonium chloride and benzethonium chloride are each useful in radiopharmaceutical preparations as bacteriostatic agents which are compatible with anti-oxidants.

SUMMARY OF THE INVENTION

The present invention is related to the use of benzalkonium chloride orbenzethonium chloride as bacteriostatic agents in radiopharmaceuticalpreparations.

This invention also relates to radiopharmaceutical compositionscomprising benzalkonium chloride or benzethonium chloride and aradiopharmaceutical agent.

BRIEF DESCRIPTION OF DISCLOSURES IN THE ART

Benzalkonium chloride has been widely used as a preservative forpharmaceutical preparations, especially in ophthalmic, dermatologic,gynecological and dental applications. For example, a European PatentApplication (EPO Publication No. 0,306,984) and a publication (J. Pharm.Pharmac., 1972, 24, 145-148) describe the use of benzalkonium chloridein ophthalmic formulations. Benzalkonium chloride may also be utilizedin a composition for the cleaning and storage of contact lenses (seeU.S. Pat. No. 3,882,036 and Japanese Patent Application JP 62/153217).Benzalkonium chloride has also been used as a preservative forpharmaceutical compositions for nasal administration (see EPOPublication No. 0,193,372 and UK Patent Application GB 2,127,689A).

Benzethonium chloride may be utilized in similar applications. Due toits low water solubility, benzethonium chloride has been primarilyutilized in antiperspirant and deodorant sticks.

BACKGROUND OF THE INVENTION

Noninvasive, nuclear imaging techniques can be used to obtain basic anddiagnostic information about the physiology and biochemistry of avariety of living subjects including experimental animals, normalhumans, and patients. These techniques rely on the use of sophisticatedimaging instrumentation which is capable of detecting radiation emittedfrom radiotracers administered to such living subjects. The informationobtained can be reconstructed to provide planar and tomographic imageswhich reveal the distribution of the radiotracer as a function of time.Use of appropriately designed radiotracers can result in images whichcontain information on structure (low resolution), function, and mostimportantly, physiology and biochemistry of the subject. Much of thisinformation cannot be obtained by any other means. The radiotracers usedin these studies are designed to have defined behaviors in vivo whichpermit the determination of specific information concerning thephysiology or biochemistry of the subject or of the effect that variousdiseases or drugs have on the physiology or biochemistry of the subject.Currently, radiotracers are available for obtaining useful informationconcerning such things as cardiac function, myocardial blood flow, lungperfusion, bone density, liver function, kidney function, brain bloodflow, regional brain glucose, and oxygen metabolism.

A variety of radiotracers have been proposed for radioimaging includingcompounds labeled with either positron or gamma emitting nuclides. Forimaging, the most commonly used positron emitting radiotracers are ¹¹ C,¹⁸ F, ¹⁵ O, and ¹³ N, all of which are accelerator produced, and havehalf-lives of 20, 110, 10, and 2 min respectively. Since the half-livesof these radionuclides are so short, it is only feasible to use them atinstitutions which have an accelerator on site for their production,limiting their use to approximately 25 medical centers in the U.S. andonly about 50 throughout the world. Several gamma emitting radiotracersare available which can be used by essentially any hospital in the U.S.and in most hospitals throughout the world. The most widely used ofthese are ^(99m) Tc (Tc-99m), ²⁰¹ Tl, ¹²³ I and ¹³¹ I. ²⁰¹ Tl is amonovalent cation which is used for measuring myocardial blood flow.Both ^(99m) Tc and ¹³¹ I can be incorporated into a variety ofradiotracers and are widely used in most modern hospitals. ^(99m) Tc isgenerator produced, has a 6 hour half-life, and emits a 140 keV gammaphoton which makes this radionuclide nearly ideal for use with currentplanar and single photon emission computerized tomography (SPECT)cameras. ^(99m) Tc is a transition metal which forms a wide variety ofcomplexes with molecules containing coordinating ligands (e.g. moleculeswith free thiol, amine, carboxyl or phosphonate functional groups).^(99m) Tc labeled compounds have been developed for many diagnosticimaging applications, such as functional studies (e.g. cardiac, renal,liver) and perfusion studies (e.g. myocardial, brain, lung, bone).

Diagnostic imaging kits which employ technetium-99m generally compriseseveral components, i.e. a source of Tc-99m, a ligand, a reducing agentand an antioxidant. The diagnostic agent is generally formed by reactingTc-99m in an oxidized state with an appropriate ligand in the presenceof a reducing agent under conditions which allow formation of a stablecomplex between Tc-99m in a reduced state (e.g., III, IV or V valencestate) and the ligand. The complex should have the desired property ofbecoming localized in the target organ upon introduction into a patient.Additionally, an antioxidant may be present to suppress the formation ofunwanted impurities from the reduction.

To facilitate handling and storage, the aforementioned components of^(99m) Tc-based imaging kits are generally kept in a freeze-dried stateprior to reconstitution. Such lyophilized components may packagedindividually or in various combinations as warranted by the specificapplication. Prior to administration the components of the kit arereconstituted by the addition of sodium pertechnetate in saline andmixing, if separately packaged. The shelf life of lyophilized ^(99m)Tc-based radiopharmaceuticals prior to reconstitution may be as long as12 to 18 months. Upon reconstitution, however, the shelf life is only 6hours. Because many hospitals generally make up a single large batch ofinjection solution, the short post-reconstitution shelf life imposesserious constraints on efficient management of diagnostic procedures.

Such a short post-reconstitution shelf life is due to regulationsconcerning bacterial growth in the parenteral solutions. Although a widevariety of antibacterial agents are known in the art, very few have beenutilized in radiopharmaceutical preparations. This is primarily due totheir incompatibility with antioxidants. For example methylparaben andpropylparaben are utilized as antibacterial agents in commerciallyavailable ^(99m) Tc-based radiopharmaceuticals. Because they areincompatible with the antioxidant employed in the composition, theconcentration of ^(99m) Tc complex must be kept low to avoid theformation of undesireable by-products. The ^(99m) Tc-based by-productsdiminish the quality and resolution of diagnostic imaging. Otherbacteriostats, including phenol, thymol, benzyl alcohol, and phenylethylalcohol, are similarly imcompatible with antioxidants.

Benzalkonium chloride is known as a topical antiinfective, antisepticand antimicrobial agent. Benzalkonium chloride is bacteriostatic in lowand bactericidal in high concentrations. Gram-positive bacteria are moresensitive than gram-negative bacteria. Benzalkonium chloride is used forapplication to skin and mucous membranes. It is widely used inover-the-counter ophthalmic solutions and in compositions for cleaningand storing contact lenses. It is also used for the sterilization ofinanimate articles, such as surgical instruments. For a review of theantimicrobial activity and use of benzalkonium chloride see W. Gump,"Disinfectants and Antiseptics" in Kirk-Othmer Encyclopedia of ChemicalTechnology, Vol. 7 (Wiley-Interscience, New York, 3rd. ed; 1979) pp.815-818.

Benzethonium chloride has similar properties, but, due to its low watersolubility, it is primarily utilized for cosmetic applications and inantiperspirant and deodorant sticks.

As noted earlier, all current Tc-99m radiopharmaceuticals require theaddition of an anti-oxidant and a bacteriostat to extend shelf life postreconstitution. Presently, bacteriostats are seldom used because theyinterfere with most anti-oxidants and thus the shelf life is limited byregulations to six hours.

Accordingly, an object of the present invention is to provide abacteriostatic agent for radiopharmaceutical preparations which iscompatible with anti-oxidants.

It has now been surprisingly found that radiopharmaceutical compositionscan be obtained wherein both bacterial growth and oxidation areminimized. The present invention has met this need by being compatiblewith current anti-oxidants, allowing the extension of the shelf life upto 24 hours post reconstitution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to radiopharmaceutical compositionscomprising:

(a) a ^(99m) Tc-based radiopharmaceutical;

(b) a water-soluble pertechnetate reducing agent;

(c) a radical-scavenging antioxidant; and

(d) a bacteriostat selected from:

(i) benzalkonium chloride, and

(ii) benzethonium chloride.

The present invention is also directed to methods of utilizingradiopharmaceutical formulations comprising:

(a) a ^(99m) Tc-based radiopharmaceutical;

(b) a water-soluble pertechnetate reducing agent;

(c) a radical-scavenging antioxidant; and

(d) a bacteriostat selected from:

(i) benzalkonium chloride, and

(ii) benzethonium chloride for diagnostic imaging (including bonescintigraphy, kidney radioimaging, lung radioimaging, brainradioimaging, and blood pool and myocardial infarct radioimaging).

As used herein, a "^(99m) Tc-based radiopharmaceutical" is a complex of^(99m) Tc in the III, IV or V oxidation state with an organic ligand(s).Such radiopharmaceutical may be pre-formed prior to combination withother ingredients or may be formed in situ.

The source of Tc-99m should preferably be water soluble. Preferredsources are alkali and alkaline earth metal salts of pertechnetate (TcO₄⁻) such as, for example, sodium pertechnetate. Tc-99m is most preferablyobtained in the form of fresh sodium pertechnetate from a sterile Tc-99mgenerator (e.g., from a conventional ⁹⁹ Mo/^(99m) Tc generator). Anyother source of physiologically acceptable Tc-99m may be used.

Tc-99m in the form of pertechnetate can be used in amounts up to about200 mCi/ml, preferably between about 20-200 mCi/ml.

The ligand or chelating agent for ^(99m) Tc which is selected will varywith the desired diagnostic imaging application. Such ligand shouldcomplex quickly with ^(99m) Tc to form a stable, biologically acceptablecomplex.

For the preparation of kits for bone scintigraphy, the preferred ^(99m)Tc ligand is a diphosphonic acid, pyrophosphate or trimetaphosphate. Forthe preparation of kits for bone scintigraphy it is even more preferredthat the diphosphonic acid be selected from: methylene-diphosphonic acid(MDP), hydroxymethylene-diphosphonic acid (HMDP),ethane-1-hydroxy-1,1-diphosphonic acid (EHDP),1-hydroxypropane-1,1-diphosphonic acid,2,3-dicarboxypropane-1,1-diphosphonic acid, pyrophosphate ortrimetaphosphate.

For the preparation of kits for radioimaging of the kidney, thepreferred ^(99m) Tc ligand is selected from:diethylenetriaminepentaacetic acid (pentetate)(N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-glycine),2,3-dimercaptosuccinic acid (DMSA), glycero-glucoheptanoic acid, orN-[N-[N-(mercaptoacetyl)glycyl]glycyl]glycine.

For the preparation of kits for radioimaging of the lung, the preferred^(99m) Tc ligand is selected from: macroaggregated albumin (MAA),aggregated human albumin, albumin colloid, or recombinant humanalbumin/human serum albumin in microspherical form.

For the preparation of kits for blood pool and myocardial infarctimaging, the preferred ^(99m) Tc ligand is selected from:iminodiphosphonic acid, [(acac)₂ en], hydrophosphonic acid,glyceroglucoheptanoic acid, a monoclonal antibody (MAb), especially amonoclonal antibody to fibrin or to myosin, tissue plasminogen activator(tPA), 1-isocyano-2-methoxy-2-methylpropane (sestamibi), or[tris(1,2-cyclohexanedionedioximato)-O]-methylborane (teboroxime).

For the preparation of kits for brain/cerebral perfusion imaging thepreferred ^(99m) Tc ligand is selected from: diethylenetriaminepentaacetic acid,[tris(2,3-butanedione-dioximato)-O]-(2-methylpropyl)borane (siboroxime),ethyl cystinate dimer (neurolyte), or3,3'-[(2,2-dimethyltrimethylene)diimino]di-2-butanone-dioxime(exametazime).

For the preparation of kits for radioimaging of the liver, gall bladder,and/or spleen the preferred ^(99m) Tc ligand is selected from:N-[2-[(3-bromo-2,4,6-trimethylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)-glycine(mebrofenin),N-[2-[[2,6-bis(1-methylethyl)phenyl]amino]-2-oxoethyl]-N-(carboxymethyl)-glycine(disofenin),N-(carboxymethyl)-N-[2-[2,6-dimethylphenyl)-amino]-2-oxoethyl]glycine(lidofenin), or polyisohexylcyanoacrylate nanoparticles.

For the preparation of kits for radioimaging of tumors in breast,colorectal, ovarian, pancreatic, prostate, small-cell lung, and/ornon-small-cell lung cancers and non-Hodgkin's lymphoma the preferred^(99m) Tc ligand is selected from: a monoclonal antibody (MAb),especially a monoclonal antibody selected from the group consisting of:LS2D617, NR-LU-10, 28A32, 703D4 (ATCC HB 8301), 704A1 (ATCC HB 8302),murine IgG1 monoclonal antibody BTMA8, or a monoclonal antibody from ahuman myeloma cell line.

The amount of the ligand may range from about 0.5 mg/ml up to the amountmaximally soluble or suspendable in the medium.

The pharmaceutically acceptable salts of the radiopharmaceuticalsinclude the conventional non-toxic formed, e.g., from non-toxicinorganic or organic acids or bases. For example, such conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic,and the like. Base salts include ammonium salts, alkali metal salts suchas sodium and potassium salts, alkaline earth metal salts such ascalcium an magnesium salts, salts with organic basis such asdicyclohexylamine salts, N-methyl-D-glucamine, and salts with aminoacids such as arginine, lysine, and so forth.

The "radical scavenging antioxidant" is an organic compound whichprevents or inhibits the re-oxidation of reduced technetium-99m topertechnetate (Tc VII) by diminishing the effect of dissolved oxygen inthe formulation. Suitable radical scavenging antioxidants includepara-aminobenzoic acid (PABA), gentisic acid, and ascorbic acid. Theradical scavenging antioxidant is generally present in amounts of about0.01-10 mg/ml.

Reducing agents must be physiologically acceptable and effective forreducing technetium-99m from its oxidized state to the III, IV or Voxidation state. In the present invention, the choice of reducing agentis not critical, but it is preferred that it be water-soluble. As usedherein the term "pertechnetate reducing agent" is intended to includecompounds, complexes, or the like, comprising a reducing ion capable ofreducing heptavalent technetium (TcO₄ ⁻) to trivalent, tetravalentand/or pentavalent technetium. Free metals such as tin are also knownfor use as pertechnetate reducing agent. Thus, it is more convenient touse metal compounds which provide the reducing metal cation in solubleform. Examples of suitable water-soluble reducing agents are stannouschloride, stannous fluoride, stannous tartrate, and sodium dithionite.The preferred agents are stannous reducing agents, such as stannouschloride and stannous fluoride. The most preferred agent is stannouschloride.

The amount of reducing agent used is the amount necessary to reduce thetechnetium to provide for binding to the ligand in a reduced state. In apreferred mode, stannous chloride (SnCl₂) is the reducing agent, and theconcentration may range between about 1-5000 μg/ml, preferably betweenabout 30-500 μg/ml.

As used herein the term "bacteriostat" includes chemical agents whichinhibit bacterial growth. At higher concentrations the instantcompositions exhibit bacteriolytic activity (i.e. cause the destructionor dissolution of bacterial cells) and/or bacteriocidal activity (i.e.kill bacteria). Such compositions and utilities are also encompassedwithin the scope of the instant invention.

Apart from applications to ^(99m) Tc-based radiopharmaceuticals, thepresent invention has utility in all radiopharmaceuticals which requirereconstitution. Additionally, the present invention contemplatesbenzalkonium chloride or benzethonium chloride as a bacteriocide inradiopharmaceutical solutions which do not require reconstitution,especially those comprising a radiohalogen, in particular ¹²³ I or ¹³¹I.

Accordingly, an alternate embodiment of the present invention isdirected to radiopharmaceutical composition comprising:

(a) a radioactive iodine-based radiopharmaceutical;

(b) an autoradiolytic decomposition-inhibiting antioxidant selectedfrom:

(i) ascorbic acid

(ii) nicotinamide,

(iii) nicotinic acid, and

(iv) a mixture of acorbic acid and nicotinamide;

(c) a bacteriostat selected from:

(i) benzalkonium chloride, and

(ii) benzethonium chloride.

The alternate embodiment of the present invention is also directed tomethods of utilizing radiopharmaceutical formulations comprising:

(a) a radioactive iodine-based radiopharmaceutical;

(b) an autoradiolytic decomposition-inhibiting antioxidant selectedfrom:

(i) ascorbic acid

(ii) nicotinamide,

(iii) nicotinic acid, and

(iv) a mixture of acorbic acid and nicotinamide;

(c) a bacteriostat selected from:

(i) benzalkonium chloride, and

(ii) benzethonium chloride

for diagnostic imaging (including bone scintigraphy, kidneyradioimaging, lung radioimaging, brain radioimaging, and blood pool andmycardial infarct radioimaging).

As used herein, a "radioactive iodine-based radiopharmaceutical" is aradiotracer wherein ¹²² I, ¹²³ I, ¹²⁵ I or ¹³¹ I is incorporated into anorganic or inorganic molecule.

For the preparation of radiopharmaceutical formulations for diagnosticimaging it is preferred that the radioactive iodine-basedradiopharmaceutical is selected from the group consisting of: [¹²³ I],[¹²⁵ I], or [¹³¹ I]-N-[2-iodo-benzoyl]-glycine (ortho-iodohippurate);[¹³¹I]N,N,N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propanediamine;[¹²³I]1,3-dihydro-3-(4-iodobenzoylamino)-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one;[¹²³I]N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzoiazepin-3-yl)-N'-(3-iodophenyl)urea;[¹²³ I]4-iodo-alphamethyl-N-(1-methylethyl)benzeneethanamine; [¹³¹I]-19-iodo-cholest-5-en-3β-ol; [¹²³ I] or [¹³¹I]-6-iodo-cholest-5-en-3β-ol; [¹²³ I] or [¹³¹ I]-m-iodobenzylguanidine;[¹²³ I] or [¹³¹ I]-p-iodo-N-isopropyl-amphetamine; [¹²³ I] or [¹³¹I]-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide;[¹²³ I] or [¹³¹ I]-9-(3,3-diethylureido)-4,6,6a,7,8,9-hexahydro-7-methyl-2'-ioddoindolo[4,3-f,g]quinoline(iodo-lisuride); [¹²³ I] or [¹³¹I]-N-(8-benzyl-1αα,5αH-nortropan-3β-yl)2,3-dimethoxy-4-iodo-benzylamide(iodo-tropapride); [¹²³ I] or [¹³¹I]-N-(2-diethylaminoethyl)-4-iodobenzamide.

As used herein, an "autoradiolytic decomposition-inhibiting antioxidant"is an organic compound which diminishes the chemical breakdown of theradiopharmaceutical induced by radioactive decay of the radioactivespecies itself. Suitable autoradiolytic decomposition inhibitingantioxidants including ascorbic acid, nitotinamide, nicotinic acid, orcombinations thereof. The use of such antioxidants is described in U.S.Pat. No. 4,880,615. These compounds retard the radiolytic decompositoncaused by radioactive decay of iodine thereby prolonging their usefulshelf-life. The autoradiolytic decomposition inhibiting antioxidant isgenerally present in amounts of about 0.01-20 mg/ml.

Although both benzalkonium chloride (BAC) and benzethonium chloride(BEC) have utility in the present invention, benzalkonium chloride ispreferred. Benzalkonium chloride (N-alkylbenzyldimethylammonium chlorideand/or N-dialkylbenzylmethylammonium chloride) (C.A.S. Registry No.8001-54-5) is commercially available, or may be prepared by methods wellknown in the art.

Benzalkonium chloride is a mixture of N-alkylbenzyldimethylammoniumchlorides of the general formula [C₆ H₅ CH₂ N(CH₃)₂ R]Cl, in which Rrepresents a mixture of alkyls, including all or some of the groupbeginning with n--C₈ H₁₇ and extending through higher homologs of n--C₂₀H₄₁, with n--C₁₂ H₂₅, n--C₁₄ H₂₉ and n--C₁₆ H₃₃ comprising the majorportion. In general, on the anhydrous basis, the content of n--C₁₂ H₂₅homolog is not less than 30%, and the content of the n--C₁₄ H₂₉ homologis not less than 10%, of the total alkylbenzyldimethyl-ammonium chloridecontent. The amounts of the n--C₁₂ H₂₅ and n--C₁₄ H₂₉ homolog componentsgenerally comprise together not less than 50% of the totalalkylbenzyldimethylammonium chloride content. Also contemplated withinthe scope of the present invention are benzalkonium chloride species ofthe general formula [C₆ H₅ CH₂ N(CH₃)RR]Cl, in which R is independentlyas defined above. Such N-alkyldimethylbenzylammonium chloride andN-dialkylmethylbenzylammonium chloride species may be presentindividually or together in a ratio of 10:1 to 1:10. Commerciallyavailable benzalkonium chloride may compriseN-alkyldimethylbenzylammonium chlorides alone, or may additionallycomprise N-dialkylmethylbenzylammonium chlorides in the aforementionedratios.

Although it is preferred that the benzalkonium species be in thechloride salt form (i.e. benzalkonium chloride), other salt forms arealso contemplated within the scope of the instant invention, with thebromide, iodide, acetate, benzoate, sulfonate, benzenesulfonate, andphosphonate being exemplary.

The ^(99m) Tc-based radioactive diagnostic agent or the non-radioactivecarrier therefore may be formulated in a lyophilized composition, asimple powdery composition, an aqueous solution or the like. In additionto the said essential components, it may contain any conventionaladditive, such as a pH-adjusting agent and an isotonizing agent (e.g.sodium chloride).

The radioiodine-based radioactive diagnostic agent or thenon-radioactive carrier therefore is generally formulated in an aqueoussolution. In addition to the said essential components, it may containany conventional additive, such as a pH-adjusting agent and anisotonizing agent (e.g. sodium chloride).

For diagnostic imaging in a subject the radiopharmaceutical compositionsof the present invention may be adminstered by intravenous, oral, ornebular administration. For bone scintigraphy in a subject, intravenousadministration is preferred. For radioimaging of the kidney in asubject, intravenous administration is preferred. For radioimaging ofthe lung in a subject, intravenous or nebular admministration ispreferred. For radioimaging of blood pool and myocardial infarct in asubject, intravenous administration is preferred. For radioimaging ofthe brain in a subject, intravenous administration is preferred. Forradioimaging of the liver, gall bladder, and/or spleen in a subject,intravenous administration is preferred. For radioimaging of tumors in asubject, intravenous administration is preferred.

The concentration of the bacteriostat in the instant radiopharmaceuticalcompositions will vary with a variety of factors including the ^(99m)Tc-based radiopharmaceutical, the pertechnetate reducing agent, and theantioxidant employed, temperature and time of storage, and the specificdiagnostic application. For benzalkonium chloride the concentration ispreferably from about 0.00001% to about 0.01% (w/v), and more preferablyfrom about 0.00005% to about 0.001% (w/v), and even more preferably fromabout 0.001% to about 0.0005% (w/v). For benzethonium chloride theconcentration is preferably from about 0.00001% to about 0.01% (w/v),and more preferably from about 0.00005% to about 0.001% (w/v), and evenmore preferably from about 0.0001% to about 0.0005% (w/v).

Similarly, the concentration of the bacteriostat in theradiopharmaceutical compositions of the alternate embodiment of thepresent invention will vary with a variety of factors including theradioiodine-based radiopharmaceutical, the autoradiolyticdecomposition-inhibiting antioxidant employed, temperature and time ofstorage, and the specific diagnostic application. For benzalkoniumchloride the concentration is preferably from about 0.00001% to about0.01% (w/v), and more preferably from about 0.00005% to about 0.001%(w/v). For benzethonium chloride the concentration is preferably fromabout 0.00001% to about 0.01% (w/v), and more preferably from about0.00005% to about 0.001% (w/v).

The following examples are given for the purpose of illustrating thepresent invention and shall not be construed as being limitations on thescope or spirit of the instant invention.

EXAMPLE 1 MDP/PABA with Benzalkonium Chloride as Preservative

A preliminary radiolabelling test of benzalkonium chloride (BAC) withsodium pertechnetate ^(99m) Tc in the presence of para-aminobenzoic acid(PABA) was conducted.

Three vials with a total volume of 10 ml from a recently manufacturedmethylene diphosphonic acid (MDP) kit containing PABA were treated withvarious amounts of benzalkonium chloride, i.e., 1, 5 and 10 mg whichcorresponded to 0.01, 0.05 and 0.10% solutions. Radiochromatography wasperformed using ITLC-SG strips developed in acetone and saline. Thevials were tested at 5 and 24 hours post reconstitution. At these twoperiods, less than 1% free pertechnetate and reduced hydrolyzedtechnetium was found in the three vials.

This test demonstrated that benzalkonium chloride did not have adeteriorating effect on PABA nor on the physical appearance of theproduct (no precipitate was observed). Hence, BAC does not interferewith the anti-oxidant properties of PABA. In contrast, benzethoniumchloride caused the phosphonate to precipitate giving a cloudy solution.Benzethonium chloride, similarly, did not interfere with theanti-oxidant properties of PABA.

EXAMPLE 2 MDP/PABA Formulated with Benzalkonium Chloride-Stress Test

Four vials of MDP/PABA were reconstituted with 500 mCi of pertechnetate^(99m) Tc in 9 mL of normal saline. Immediately to two vials were added1 mg/1 mL (0.01%) of benzalkonium chloride solution. Similarly, to theremaining two vials we added 10 mg/1 mL (0.1%). The radiochromatographytests as described in Example 1 were performed at 5 and 24 hours postreconstitution (PR):

    ______________________________________                                        Percent of free .sup.99m Tc Pertechnetate                                                0.01%           0.1%                                               Time PR      #1     #2         #3   #4                                        ______________________________________                                        5 hrs        1.8    1.5        1.0  1.2                                       24 hrs       <1     <1         <1   <1                                        ______________________________________                                    

The above data shows that even 10 mg of benzalkonium chloride added with500 mCi pertechnetate ^(99m) Tc did not affect the stability of theproduct. The four solutions remained clear with no sign of precipitateup to 48 hours post labelling. Thus, even under strenuous conditions andin the presence of BAC, the antioxidant properties of PABA aremaintained.

EXAMPLE 3 MDP/PABA Compared with MDP/PABA/BAC-BIODISTRIBUTION STRESSTEST

A lyophilized batch of MDP/PABA formulated with benzalkonium chloride(MDP/PABA/BAC) was prepared and tested. The bacterial preservative wasadded prior to freeze-drying. No special precaution was taken in orderto avoid excessive air oxidation of the [tin].

A side by side study of biodistribution of the two formulations with andwithout benzalkonium chloride was conducted.

A radiochemical "stress test" was also performed on three vials ofMDP/PABA/BAC. A "non-stressed" vial was tested simultaneously as acontrol.

Each lyophilized vial contained 10 mg of MDP, 1 mg of stannous chloridedihydrate, 2 mg of PABA and 50 μg of benzalkonium chloride (BAC). The pHwas adjusted to 7.0. When the benzalkonium chloride was added to thefinal solution, a slight haze appeared. The haze was filtered off togive a clear solution.

A. BIOLOGICAL DATA

The dosage was 83 μg MDP per rat (130-150 g). Tissue distribution wasmonitored at 20 minutes, 1 hour, 2 hours, 6 hours and 24 hourspost-reconstitution (PR).

As shown by the following data, the biodistribution of theradiopharmaceutical was not altered by the addition of BAC.

1. Blood elimination.

The elimination pattern was very similar for both formulations. Nosignificant difference was seen between the two formulations.

2. Liver elimination.

The liver elimination was slightly faster at first for MDP/PABA/BAC, andapproximately the same after three hours post injection.

3. Kidney uptake.

The kidney uptake and elimination was the same for both preparations.

4. Stomach and Proximal Intestine.

The stomach and proximal intestine also showed the same eliminationpattern. No significant difference between the two preparations.

5. Muscle uptake.

The muscle activity was the same for both preparations.

6. Bone uptake.

The total bone uptake was a few percent lower for the benzalkoniumformulation. The observed difference was probably insignificant, and wasdue to the general variability of bone uptake. This was not enough tomake a real difference on bone scintigraphy.

    ______________________________________                                                   Percent of Injected Dose Recovered                                              ANI-     ANI-     ANI-                                           ORGAN        MAL #1   MAL #2   MAL #3 MEAN                                    ______________________________________                                        MDP/PABA     (20 Min PR)                                                      BLOOD        3.9      3.3      3.0    3.4                                     LIVER        1.1      0.7      1.3    1.0                                     KIDNEYS      2.5      4.3      1.5    2.8                                     STOMACH + PI 0.6      1.4      0.8    0.9                                     MUSCLE       6.2      3.9      4.0    4.7                                     BONE         58.6     48.6     53.2   53.4                                    MDP/PABA     (1 Hr PR)                                                        BLOOD        0.3      0.3      0.3    0.3                                     LIVER        0.3      0.6      0.5    0.5                                     KIDNEYS      0.7      0.8      0.9    0.8                                     STOMACH + PI 0.2      0.1      0.1    0.1                                     MUSCLE       0.5      0.6      0.5    0.1                                     BONE         65.6     64.5     63.8   64.8                                    MDP/PABA     (3 Hr PR)                                                        BLOOD        0.1      0.1      0.1    0.1                                     LIVER        0.1      0.1      0.1    0.1                                     KIDNEYS      0.5      0.1      0.4    0.3                                     STOMACH + PI 0.1      0.1      0.1    0.1                                     MUSCLE       0.7      0.6      0.6    0.7                                     BONE         45.6     48.0     48.6   47.4                                    MDP/PABA     (6 Hr PR)                                                        BLOOD        0.1      0.1      0.1    0.1                                     LIVER        0.1      0.1      0.1    0.1                                     KIDNEYS      0.5      0.6      0.5    0.5                                     STOMACH +  PI                                                                              0.1      0.1      0.1    0.1                                     MUSCLE       0        0        0      0                                       BONE         55.0     54.5     49.4   53.0                                    MDP/PABA     (24 Hr PR)                                                       BLOOD        0.1      0.1      0.1    0.1                                     LIVER        0.2      0.4      0.4    0.3                                     KIDNEYS      0.5      0.8      0.7    0.7                                     STOMACH + PI 0.02     0.02     0.02   0.02                                    MUSCLE       0.2      3.5      0.2    1.3                                     BONE         60.6     63.8     71.4   65.2                                    MDP/PABA/BAC (20 Min PR)                                                      BLOOD        2.1      1.7      2.5    2.1                                     LIVER        0.5      0.3      0.4    0.4                                     KIDNEYS      1.1      2.2      1.2    1.5                                     STOMACH + PI 0.3      0.2      0.3    0.3                                     MUSCLE       2.3      0.1      0.7    1.0                                     BONE         49.3     43.2     54.2   48.9                                    MDP/PABA/BAC (1 Hr PR)                                                        BLOOD        0.3      0.3      0.3    0.3                                     LIVER        0.1      0.1      0.1    0.1                                     KIDNEYS      0.5      0.5      0.9    0.6                                     STOMACH + PI 0.2      0.2      0.2    0.2                                     MUSCLE       0.6      0.5      0.5    0.5                                     BONE         59.4     57.8     54.8   57.3                                    MDP/PABA/BAC (3 Hr PR)                                                        BLOOD        0.2      0.1      0.1    0.1                                     LIVER        0.6      0.1      0.1    0.3                                     KIDNEYS      0.1      0.5      0.5    0.4                                     STOMACH + PI 0.1      0.1      0.1    0.1                                     MUSCLE       0.1      0.3      0.2    0.2                                     BONE         54.8     48.8     60.1   54.6                                     MDP/PABA/BAC                                                                              (6 Hr PR)                                                        BLOOD        0.1      0.1      0.1    0.1                                     LIVER        0.1      0.1      0.1    0.1                                     KIDNEYS      0.5      0.6      0.6    0.6                                     STOMACH + PI 0.1      0.3      1.2    0.5                                     MUSCLE       0.1      0.1      0.1    0.1                                     BONE         49.1     63.5     55.0   55.9                                    MDP/PABA/BAC (24 Hr PR)                                                       BLOOD        0.1      0.1      0.1    0.1                                     LIVER        0.1      0.1      0.1    0.1                                     KIDNEYS      0.5      0.5      0.2    0.4                                     STOMACH + PI 0.1      0.1      0.1    0.1                                     MUSCLE       0.2      0.4      0.3    0.3                                     BONE         55.0     55.9     47.5   52.8                                    ______________________________________                                    

B. RADIOCHEMICAL "STRESS TEST"

Four vials of the lyophilized product were labelled with ^(99m)Tc-sodium pertechnetate. Each received 500 mCi in 10 mL of technetiumfrom a recent elution. For the first three vials, twenty minutes afterreconstitution, 7 mL were removed. The fourth vial remained untouched.The vials were kept at room temperature throughout the length of theexperiment.

The radiochemical tests were performed at 0.5, 6.0 and 24 hours postreconstitution. At all times, the unbound pertechnetate remained lessthan 1%.

EXAMPLE 4

A test of the bacteriostatic effects of various concentrations ofbenzalkonium chloride in MDP/PABA was conducted. A total of fourteenvials, two each of the six concentrations of benzalkonium chloride inMDP/PABA and two control MDP/PABA vials were utilized. A dilute sourceof microorganisms was prepared by adding 3 drops of a "dust culture"(which was a mixed culture derived from dust and comprised a variety ofwild strains of bacteria and fungi typical to ambient conditions) to 1liter of sterile water for injection. After sufficient agitation, threedrops of the dilute culture were added to one vial of eachconcentration, including one zero vial, and all fourteen vials were leftto incubate overnight (approx. 18 hours) at room temperature. One tube(15 mL) of Fluid Thioglycollate medium and one tube (15 mL) of TrypticSoy medium was inoculated with 1 milliliter from each of the fourteenincubated vials. Each tube was incubated for fourteen days. At the endof the normal fourteen day incubation period, all sixteen culture tubesrepresenting concentrations of 50 μg and over (those tubes which did notshow growth) were inoculated with concentrated "dust culture" to provetheir ability to maintain growth at the end of the period.

CONCLUSION

Growth was observed in both media at benzalkonium chlorideconcentrations of 10 μg and 25 μg per vial, while concentrations ofgreater than 25 μg per vial and higher produced no growth. All controlsshowed no growth as expected. The beneficial effect of the benzalkoniumchloride was evident in the viability test at concentrations of 250 μgper vial and over.

    ______________________________________                                        RESULTS                                                                       ______________________________________                                        A - STERILITY TEST                                                            BAC          INOCULATED    CONTROL                                            CONCENTRATION                                                                              THIO.    SOY      THIO.  SOY                                     ______________________________________                                        ZERO         POS.     POS.     NEG.   NEG.                                                 1/18     1/22     1/30   1/30                                     10 μg    POS.     POS.     NEG.   NEG.                                                 1/19     1/22     1/30   1/30                                     25 μg    POS.     POS.     NEG.   NEG.                                                 1/22     1/22     1/30   1/30                                     50 μg    NEG.     NEG.     NEG.   NEG.                                                 1/30     1/30     1/30   1/30                                     100 μg   NEG.     NEG.     NEG.   NEG.                                                 1/30     1/30     1/30   1/30                                     250 μg   NEG.     NEG.     NEG.   NEG.                                                 1/30     1/30     1/30   1/30                                    1000 μg   NEG.     NEG.     NEG.   NEG.                                                 1/30     1/30     1/30   1/30                                    ______________________________________                                        B - VIABILITY CHECK                                                                       DATE GROWTH OBSERVED                                                            THIO        SOY                                                 ______________________________________                                        50 μg control                                                                            2/1         2/2                                                 innoc.        2/1         2/2                                                 100 μg control                                                                           2/1         2/2                                                 innoc.        2/1         2/6                                                 250 μg control                                                                           NEG.        NEG.                                                innoc.        NEG.        NEG.                                                1000 μg control                                                                          NEG.        NEG.                                                innoc.        NEG.        NEG.                                                ______________________________________                                    

EXAMPLE 5 MDP/PABA/BAC Formulation

This test was designed to determine a suitable ratio of SnCl₂ •2H₂ O tobenzalkonium chloride that would satisfy the radiochemical "stress test"and still remain clear on reconstitution. It was also desired to producea solution which would not be colloidal prior to the freeze-dryingprocess.

Four batches of MDP/PABA/BAC kits were prepared, lyophilized, andtested. The formulations were as follows:

1. 10 mg MDP, 1.0 mg SnCl₂.2H₂ O, 2 mg PABA, 50 μg Benzalkonium Cl.

2. 10 mg MDP, 1.0 mg SnCl₂ •2H₂ O, 2 mg PABA, 25 μg Benzalkonium Cl.

3. 10 mg MDP, 0.5 mg SnCl₂ •2H₂ O, 2 mg PABA, 50 μg Benzalkonium Cl.

4. 10 mg MDP, 0.5 mg SnCl₂ •2H₂ O, 2 mg PABA, 25 μg Benzalkonium Cl.

The benzalkonium chloride was added as a dilute solution to the finalpreparations. Prior to filtration, batch #1 was found to be slightlycolloidal. Batch #3 was also colloidal, but to a much lesser degree.Batches #2 and #4, which had less benzalkonium chloride, were visiblyclear prior to filtration. All solutions were clear after the finalfiltration through a 0.22 μm membrane filter. Therefore, the morebenzalkonium chloride in solution, the more precipitation observed priorto filtration. Approximately twenty vials of each formulations werelyophilized. The results of testing performed on the final products areas follows:

A. Physical Appearance

Upon reconstitution, batches #1 and #2 were clear. Batch #3 and #4 wereslightly colloidal with low volume reconstitution (less than 2 mL). Onreconstitution with larger volumes (5 mL or more), batches #3 and #4were clear. In general, these four formulations were clearer than theMDP/PABA kit vials reconstituted with less than 3 mL per vial. In fact,most of the kits reconstituted with low volume (<3 mL) looked slightlycolloidal. Therefore, the benzalkonium chloride may actually makereconstituted vials clearer.

B. Radiochemical "Stress Test"

One vial from each batch was reconstituted with 500 mCi ^(99m) Tc-sodiumpertechnetate in 10 mL. Twenty minutes later, 7 mL were removed. Thevials were left at room temperature for the duration of the test. Thepercent reduced TcO₂ was found to be less than 1% for the whole testingperiod, thus these results were not included in the following table:

    ______________________________________                                        Percent TcO.sub.4                                                             Time         #1     #2         #3   #4                                        ______________________________________                                        0.5 Hr P-R   <1     <1         <1   <1                                        3.0 Hr P-R   <1     <1         <1   <1                                        6.0 Hr P-R   <1     <1         <1   <1                                        24 Hr P-R    <1     1.9        77.8 84.2                                      ______________________________________                                         (P-R = PostReconstitution)                                               

C. Biological Test

For each batch, three adult rats were injected, and sacrificed one hourlater. The results are expressed, below, as the mean ± the standarddeviation:

    ______________________________________                                        Percent of Injected Dose                                                      (Dosage was 83 μg/0.5 mL)                                                  ORGAN      #1        #2       #3      #4                                      ______________________________________                                        BLOOD      0.6(0.2)  0.4(0.1) 0.2(0.1)                                                                              0.5(0.2)                                LIVER      0.2(0.1)  0.2(0.1) 0.2(0.0)                                                                              0.2(0.0)                                KIDNEYS    0.7(0.1)  0.8(0.3) 0.9(0.3)                                                                              1.4(1.0)                                STOMACH + PI                                                                             0.2(0.2)  0.1(0.0) 0.2(0.1)                                                                              0.3(0.2)                                MUSCLE     0.9(0.2)  1.0(0.0) 0.8(0.2)                                                                              0.9(0.3)                                BONE       47.6(10.3)                                                                              54.5(9.5)                                                                              59.4(1.8)                                                                             56.7(5.2)                               ______________________________________                                    

Conclusion

Physically, batches #1 and #2 which have 1 mg of SnCl₂ •2H₂ O wereclearer on reconstitution with low volume than batches #3 and #4 (whichhave 0.5 mg of SnCl₂ •2H₂ O). Batch #1 was slightly cloudy. Batch #2 wasthe clearest. Batches #3 and #4 were cloudier on reconstitution with lowvolume, but no more than our regular kits.

The radiochemical "stress test" showed that 0.5 mg of SnCl₂ •2H₂ O wasinsufficient to maintain efficient labelling up to 24 hours postreconstitution.

The biological tests performed in rats do not show significantdifferences between these four batches after early reconstitution.

Overall, BAC neither improved nor diminished the reducing capacity ofstannous chloride in the radiopharmaceutical formulations.

EXAMPLE 6

A microbiological test was conducted according to USP XXI. The resultsshow that 10 or 25 μg BAC in a volume of 10 ml will provide effectivebacteriostasis by inhibiting bacterial, fungal and mold growth.

    ______________________________________                                        Trial 1: MDP Control                                                                       Original           Adjusted                                                   Inoculum  Dilution Cell Suspension                               Organism     (cfu/ml)  Needed   (cfu/ml)                                      ______________________________________                                        Aspergillis niger                                                                          1.80 × 10.sup.5                                                                   --       1.80 × 10.sup.5                         (ATCC 16404)                                                                  Candida albicans                                                                           1.56 × 10.sup.6                                                                   --       1.56 × 10.sup.6                         (ATCC 10231)                                                                  Staphyloccoccus                                                                            5.88 × 10.sup.7                                                                   1/10     5.88 × 10.sup.6                         aureus (ATCC 6538)                                                            (10 ml volume) (0.1 ml of the adjusted cell suspension were                   added to each of 20 ml of the product. Product on test was                    stored at 20° C.)                                                      ______________________________________                                                      Aspergillis                                                                              Candida  Staphylococcus                                            Niger      Albicans Aureus                                      Day           (cfu/ml)   (cfu/ml) (cfu/ml)                                    ______________________________________                                         0    Saline  1.76 × 10.sup.5                                                                    1.76 × 10.sup.6                                                                  2.08 × 10.sup.7                        0    Sample  1.60 × 10.sup.5                                                                    1.16 × 10.sup.6                                                                  1.04 × 10.sup.7                        7    Saline  1.28 × 10.sup.5                                                                    6.96 × 10.sup.5                                                                   8.0 × 10.sup.5                        7    Sample  1.44 × 10.sup.5                                                                    7.12 × 10.sup.4                                                                   9.2 × 10.sup.5                       14    Saline  0.96 × 10.sup.5                                                                    3.72 × 10.sup.5                                                                  0                                           14    Sample  1.44 × 10.sup.5                                                                    2.36 × 10.sup.3                                                                  12                                          21    Saline  0.84 × 10.sup.5                                                                    1.60 × 10.sup.5                                                                  0                                           21    Sample  1.32 × 10.sup.5                                                                    4.80 × 10.sup.3                                                                  0                                           28    Saline  1.08 × 10.sup.5                                                                    0.84 × 10.sup.5                                                                  0                                           28    Sample  1.52 × 10.sup.5                                                                    3.24 × 10.sup.3                                                                  0                                           % viable after                                                                          95%         0.28%     0                                             28 days                                                                       ______________________________________                                        Trial 2: MDP + 10 μg BAC                                                                Original           Adjusted                                                   Inoculum  Dilution Cell Suspension                               Organism     (cfu/ml)  Needed   (cfu/ml)                                      ______________________________________                                        Aspergillis niger                                                                          1.80 × 10.sup.5                                                                   --       1.80 × 10.sup.5                         (ATCC 16404)                                                                  Candida albicans                                                                           1.56 × 10.sup.6                                                                   --       1.56 × 10.sup.6                         (ATCC 10231)                                                                  Staphyloccoccus                                                                            5.88 × 10.sup.7                                                                   1/10     5.88 × 10.sup.6                         aureus (ATCC 6538)                                                            (10 ml volume) (0.1 ml of the adjusted cell suspension were                   added to each of 20 ml of the product. Product on test was                    stored at 20° C.)                                                      ______________________________________                                                      Aspergillis                                                                              Candida  Staphylococcus                                            Niger      Albicans Aureus                                      Day           (cfu/ml)   (cfu/ml) (cfu/ml)                                    ______________________________________                                         0    Saline  1.76 × 10.sup.5                                                                    1.76 × 10.sup.6                                                                  2.08 × 10.sup.7                        0    Sample  1.12 × 10.sup.5                                                                    1.24 × 10.sup.6                                                                  1.40 × 10.sup.7                        7    Saline  1.28 × 10.sup.5                                                                    6.96 × 10.sup.5                                                                   8.0 × 10.sup.5                        7    Sample  2.44 × 10.sup.2                                                                     1.2 × 10.sup.3                                                                  6                                           14    Saline  0.96 × 10.sup.5                                                                    3.72 × 10.sup.5                                                                  0                                           14    Sample  28         2.72 × 10.sup.2                                                                  0                                           21    Saline  0.84 × 10.sup.5                                                                    1.60 × 10.sup.5                                                                  0                                           21    Sample  4           9.6 × 10.sup.2                                                                  0                                           28    Saline  1.08 × 10.sup.5                                                                    0.84 × 10.sup.5                                                                  0                                           28    Sample   2.0 × 10.sup.1                                                                    3.64 × 10.sup.3                                                                  0                                           % viable after                                                                          0.018%     0.29%      0                                             28 days                                                                       ______________________________________                                        Trial 3: MDP + 25 μg BAC                                                                Original           Adjusted                                                   Inoculum  Dilution Cell Suspension                               Organism     (cfu/ml)  Needed   (cfu/ml)                                      ______________________________________                                        Aspergillis niger                                                                          1.80 × 10.sup.5                                                                   --       1.80 × 10.sup.5                         (ATCC 16404)                                                                  Candida albicans                                                                           1.56 × 10.sup.6                                                                   --       1.56 × 10.sup.6                         (ATCC 10231)                                                                  Staphyloccoccus                                                                            5.88 × 10.sup.7                                                                   1/10     5.88 × 10.sup.6                         aureus (ATCC 6538)                                                            (10 ml volume) (0.1 ml of the adjusted cell suspension were                   added to each of 20 ml of the product. Product on test was                    stored at 20° C.)                                                      ______________________________________                                                      Aspergillis                                                                              Candida  Staphylococcus                                            Niger      Albicans Aureus                                      Day           (cfu/ml)   (cfu/ml) (cfu/ml)                                    ______________________________________                                         0    Saline  1.76 × 10.sup.5                                                                    1.76 × 10.sup.6                                                                  2.08 × 10.sup.7                        0    Sample  0.92 × 10.sup.5                                                                    1.44 × 10.sup.6                                                                  2.84 × 10.sup.6                        7    Saline  1.28 × 10.sup.5                                                                    6.96 × 10.sup.5                                                                   8.0 × 10.sup.5                        7    Sample  28         1.12 × 10.sup.2                                                                  0                                           14    Saline  0.96 × 10.sup.5                                                                    3.72 × 10.sup.5                                                                  0                                           14    Sample  24         8        0                                           21    Saline  0.84 × 10.sup.5                                                                    1.60 × 10.sup.5                                                                  0                                           21    Sample  1.08 × 10.sup.2                                                                    4        0                                           28    Saline  1.08 × 10.sup.5                                                                    0.84 × 10.sup.5                                                                  0                                           28    Sample  1.76 × 10.sup.2                                                                    0        0                                           % viable after                                                                          0.19%       0%        0                                             28 days                                                                       ______________________________________                                        Trial 4: MDP + 50 μg BAC                                                                Original           Adjusted                                                   Inoculum  Dilution Cell Suspension                               Organism     (cfu/ml)  Needed   (cfu/ml)                                      ______________________________________                                        Aspergillis niger                                                                          1.80 × 10.sup.5                                                                   --       1.80 × 10.sup.5                         (ATCC 16404)                                                                  Candida albicans                                                                           1.56 × 10.sup.6                                                                   --       1.56 × 10.sup.6                         (ATCC 10231)                                                                  Staphyloccoccus                                                                            5.88 × 10.sup.7                                                                   1/10     5.88 × 10.sup.6                         aureus (ATCC 6538)                                                            (10 ml volume) (0.1 ml of the adjusted cell suspension were                   added to each of 20 ml of the product. Product on test was                    stored at 20° C.)                                                      ______________________________________                                                      Aspergillis                                                                              Candida  Staphylococcus                                            Niger      Albicans Aureus                                      Day           (cfu/ml)   (cfu/ml) (cfu/ml)                                    ______________________________________                                         0    Saline  1.76 × 10.sup.5                                                                    1.76 × 10.sup.6                                                                  2.08 × 10.sup.7                        0    Sample  0.68 × 10.sup.5                                                                     8.0 × 10.sup.5                                                                  1.36 × 10.sup.3                        7    Saline  1.28 × 10.sup.5                                                                    6.96 × 10.sup.5                                                                   8.0 × 10.sup.5                        7    Sample  0          24       0                                           14    Saline  0.96 × 10.sup.5                                                                    3.72 × 10.sup.5                                                                  0                                           14    Sample  0          8        0                                           21    Saline  0.84 × 10.sup.5                                                                    1.60 × 10.sup.5                                                                  0                                           21    Sample   7.2 × 10.sup.1                                                                    0        0                                           28    Saline  1.08 × 10.sup.5                                                                    0.84 × 10.sup.5                                                                  0                                           28    Sample   3.6 × 10.sup.1                                                                    0        0                                           % viable after                                                                          0.052%      0%        0                                             28 days                                                                       ______________________________________                                    

EXAMPLE 7 MDP/PABA/BAC Lyophilized Batch

Two batches of MDP/PABA with benzalkonium chloride were prepared andlyophilized. Two formulations of MDP/PABA/BAC (10 and 25 μg benzalkoniumchloride per vial) were prepared. This test was designed to evaluate thelyophilized product for radiolabelling (stress and non-stressed),physical appearance, reconstitution and presence of colloid. Thepreparations were made as follows: 10 mg MDP, 1 mg Sn²⁺ and 2 mg PABAper vial.

Prep #1: To 50 mL of purged Millex water was added 1 g of MDP. Next wasadded 2.5 ml of 0.6N HCl containing 100 mg of stannous chloridedihydrate. The pH was adjusted to 7.0. After 5 minutes, added 200 mg/10mL of PABA already adjusted to pH 7.0. Then was added 1.0 mg/0.1 mL of asolution of benzalkonium chloride. The pH remained at 7.0. Q.S. ed to100 mL, filtered and dispensed 1 mL per vial.

Prep #2: Repeated the same procedure as Prep #1 but used 2.5 mg/0.25 mLof the benzalkonium chloride solution.

After lyophilization, the two formulations reconstituted well with nosign of colloid. A solid white plug was obtained. The radiochemicallabelling was performed on two vials from each formulation. One vial wasreconstituted with 200 mCi/5 mL, the other was reconstituted with 200mCi/(10-7) mL. The same was repeated for the second formulation. Thevials were kept at room temperature for the length of the test. Theradiolabelling results are tabulated in the following table:

    ______________________________________                                        Percent free Pertechnetate (Room Temperature)                                        10 μg      25 μg                                                        Benzalkonium C1                                                                             Benzalkonium C1                                          Time PR  Regular  Stressed   Regular                                                                              Stressed                                  ______________________________________                                        0.5 Hr   <1       <1         <1     <1                                        4.0 Hr   <1       <1         <1     <1                                        24 Hr    <1       <1         <1     <1                                        ______________________________________                                    

In conclusion, both formulations showed excellent stability, even after24 hours.

EXAMPLE 8 MDP/PABA/BAC Stability of Lyophilized Batches

Two MDP/PABA batches were prepared containing 10 μg and 25 μgrespectively, of benzalkonium chloride (see Example 7). The formulationswere examined in an accelerated stability study which was conductedaccording to FDA guidelines. The two batches were kept at both: 22° C.and ambient relative humidity, and at 40° C. and 75% relative humidityfor 1 month. The accelerated conditions (40° C. and 75% relativehumidity for 1 month) correspond to 8 months under ambient conditions.

Labelling Efficiency

The stress test for labelling efficiency was performed on three vialsfrom each batch, which were stored at 40° C. and 75% R.H.

The labelling test was performed at 30 minutes and at 24 hourspost-reconstitution. From 30 minutes to 24 hours, for all three vials ofeach batch tested, there was <1% free pertechnetate, and <1% reducedhydrolyzed technecium detected.

Biodistribution

Biodistribution tests were performed at 30 minutes and 24 hourspost-labelling for each batch of MDP/PABA/BAC.

Standard MDP protocol for biotests was followed. Results were asfollows:

    ______________________________________                                                  ANI-   ANI-     ANI-     AVER-                                                MAL #1 MAL #2   MAL #3   AGE                                        ______________________________________                                        10 μg BAC                                                                              % ID/ORGAN (30 Min. Post-Labelling)                               BLOOD       0.3      0.3      0.3    0.3                                      LIVER       0.1      0.1      0.1    0.1                                      KIDNEYS     0.6      0.6      0.4    0.5                                      STOMACH + PI                                                                              0.1      0.5      0.1    0.2                                      MUSCLE      0.5      0.6      0.5    0.5                                      BONE        62.3     55.6     58.0   58.6                                     10 μg BAC                                                                              % ID/ORGAN (24 hours Post-Labelling)                              BLOOD       0.3      0.4      0.3    0.3                                      LIVER       0.1      0.2      0.1    0.1                                      KIDNEYS     1.0      0.6      0.4    0.7                                      STOMACH + PI                                                                              0.1      0.2      0.1    0.1                                      MUSCLE      1.1      1.3      0.8    1.1                                      BONE        65.6     59.2     51.1   58.6                                     25 μg BAC                                                                              % ID/ORGAN (30 Min. Post-Labelling)                               BLOOD       0.3      0.3      0.3    0.3                                      LIVER       0.1      0.1      0.1    0.1                                      KIDNEYS     0.4      0.5      0.4    0.4                                      STOMACH + PI                                                                              0.1      0.1      0.1    0.1                                      MUSCLE      0.2      0.4      0.3    0.3                                      BONE        55.4     59.1     54.8   56.5                                     25 μg BAC                                                                              % ID/ORGAN (24 hours Post-Labelling)                              BLOOD       0.1      0.2      0.6    0.3                                      LIVER       0.1      0.1      0.1    0.1                                      KIDNEYS     0.5      0.4      0.4    0.4                                      STOMACH + PI                                                                              0.1      0.1      0.1    0.1                                      MUSCLE      0.5      0.6      0.5    0.5                                      BONE        60.2     44.4     59.3   54.6                                     ______________________________________                                    

The MDP/PABA/BAC vials which had been stored for one month, at 40° C.and 75% relative humidity, performed normally (even after roomtemperature storage for 24 hours post-reconstitution). This acceleratedstability study demonstrated that benzalkonium chloride does notdiminish the shelf life of unreconstituted products.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe casual variations, adaptations, modifications, deletions, oradditions of procedures and protocols described herein, as come withinthe scope of the following claims and its equivalents.

What is claimed is:
 1. A radiopharmaceutical composition comprising:(a)a ^(99m) Tc-based radiopharmaceutical; (b) a water-soluble pertechnetatereducing agent; (c) a radical-scavenging antioxidant; and (d) abacteriostat selected from:(i) benzalkonium chloride, and (ii)benzethonium chloride.
 2. The radiopharmaceutical composition of claim 1wherein the bacteriostat is benzethonium chloride.
 3. Theradiopharmaceutical composition of claim 1 wherein theradical-scavenging antioxidant is selected from the group consistingof:para-aminobenzoic acid; gentisic acid; and ascorbic acid.
 4. A methodof administering a radiopharmaceutical agent to a subject, whichcomprises administering an effective amount of the radiopharmaceuticalcomposition of claim 1 by intravenous, oral, or nebular administration.5. The radiopharmaceutical composition of claim 1 wherein thebacteriostat is benzalkonium chloride.
 6. The radiopharmaceuticalcomposition of claim 5 wherein the water-soluble pertechnetate reducingagent is stannous chloride.
 7. The radiopharmaceutical composition ofclaim 6 wherein the radical-scavenging antioxidant is para-aminobenzoicacid.
 8. The radiopharmaceutical composition of claim 7 wherein the^(99m) Tc-based radiopharmaceutical is ^(99m) Tc-methylene-disphosphonicacid or a pharmaceutically acceptable salt thereof.
 9. Theradiopharmaceutical composition according to claim 1, wherein (d) ispresent in a concentration from about 0.00001% to about 0.01% (w/v). 10.The radiopharmaceutical composition according to claim 9, wherein (d) ispresent in a concentration from about 0.00005% to about 0.001% (w/v).11. The radiopharmaceutical composition according to claim 10, wherein(d) is present in a concentration from about 0.0001% to about 0.0005%(w/v).
 12. The radiopharmaceutical compostion of claim 1 wherein the^(99m) Tc-based radiopharmaceutical is selected from the groupconsisting of: ^(99m) Tc-methylene-disphosphonic acid; ^(99m)Tc-hydroxymethylene-diphosphonic acid; ^(99m)Tc-ethane-1-hydroxy-1,1-diphosphonic acid; ^(99m)Tc-1-hydroxypropane-1,1-diphosphonic acid; ^(99m)Tc-2,3-dicarboxypropane-1,1-diphosphonic acid; ^(99m) Tc-pyrophosphate;^(99m) Tc-trimetaphosphate; ^(99m) Tc-diethylenetriaminepentaaceticacid; ^(99m) Tc-2,3-dimercaptosuccinic acid; ^(99m)Tc-glycero-glucoheptanoic acid;[N-[N-[N-(mercaptoacetyl)glycyl]glycyl]glycinato(5-)-N,N',N",S]-oxo-[⁹9mTc]technetate(V); ^(99m) Tc-macroaggregated albumin; ^(99m) Tc-albumincolloid; ^(99m) Tc-aggregated human albumin; ^(99m) Tc-recombinant humanalbumin/human serum albumin in microspherical form; ^(99m)Tc-imminodiphosphonic acid; ^(99m) Tc-[(acac)₂ en]; ^(99m)Tc-hydrophosphonic acid; ^(99m) Tc-monoclonal antibody to fibrin; ^(99m)Tc-monoclonal antibody to myosin; ^(99m) Tc-tissue plasminogenactivator; hexakis(1-isocyano-2-methoxy-2-methylpropane)-[^(99m)Tc]technetium(I);bis[(1,2-cyclohexanedionedioximato)-(1-)-O]-[(1,2-cyclohexanedionedioximato)-(2-)-O]-methylborato-(2-)-N,N',N",N'",N"",N'""-chloro[^(99m)Tc]technetium (III);]bis(2,3-butanedione-dioximato)(1-)-O]-[(2,3-butanedione-dioximato)(2-)-O]-(2-methylpropyl)borato-(2)-N,N',N",N'",N"",N'""-chloro[^(99m)Tc]technetium; ^(99m) Tc-ethyl cystinate dimer; ^(99m)Tc-3,3'-[(2,2-dimethyltrimethylene)diimino]di-2-butanone-dioxime;bis[N-[2-[(3-bromo-2,4,6-trimethylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)-glycyl]-[^(99m)Tc]technetium;bis[N-[2-[[2,6-bis(1-methylethyl)phenyl]-amino]2-oxoethyl]-N-(carboxymethyl)-glycyl][^(99m)Tc]technetium;bis[-N-(carboxymethyl)-N-[2-[2,6-dimethylphenyl)-amino]-2-oxoethyl]-glycyl]-[^(99m)Tc]technetium; and ^(99m) Tc-polyisohexylcyanoacrylate nanoparticles; ora pharmaceutically acceptable salt thereof.
 13. The radiopharmaceuticalcomposition of claim 12 wherein the ^(99m) Tc-based radiopharmaceuticalis selected from: ^(99m) Tc-methylene-disphosphonic acid; ^(99m)Tc-hydroxymethylene-diphosphonic acid; ^(99m)Tc-ethane-1-hydroxy-1,1-diphosphonic acid; ^(99m)Tc-1-hydroxypropane-1,1-diphosphonic acid; ^(99m)Tc-2,3-dicarboxypropane-1,1-diphosphonic acid; ^(99m) Tc-pyrophosphate;and ^(99m) Tc-trimetaphosphate; or a pharmaceutically acceptable saltthereof.
 14. A method for bone scintigraphy in a subject, whichcomprises administering an effective amount of the radiopharmaceuticalcomposition of claim 13 by intravenous administration.
 15. Theradiopharmaceutical composition of claim 12 wherein the ^(99m) Tc-basedradiopharmaceutical is selected from: ^(99m)Tc-diethylenetriaminepentaacetic acid; ^(99m) Tc-2,3-dimercaptosuccinicacid; ^(99m) Tc-glycero-glucoheptanoic acid; and[N-[N-[N-(mercaptoacetyl)glycyl]glycyl]glycinato(5-)-N,N',N",S]-oxo-[⁹9mTc]technetate(V); or a pharmaceutically acceptable salt thereof.
 16. Amethod for radioimaging of the kidney in a subject, which comprisesadministering an effective amount of the radiopharmaceutical compositionof claim 15 by intravenous administration.
 17. The radiopharmaceuticalcomposition of claim 12 wherein the ^(99m) Tc-based radiopharmaceuticalis selected from: ^(99m) Tc-macroaggregated albumin; ^(99m) Tc-albumincolloid; ^(99m) Tc-aggregated human albumin; and ^(99m) Tc-recombinanthuman albumin/human serum albumin in microspherical form.
 18. A methodfor radioimaging of the lung in a subject, which comprises administeringan effective amount of the radiopharmaceutical composition of claim 17by intravenous or nebular administration.
 19. The radiopharmaceuticalcomposition of claim 12 wherein the ^(99m) Tc-based radiopharmaceuticalis selected from: ^(99m) Tc-imminodiphosphonic acid; ^(99m) Tc-[(acac)₂en]; ^(99m) Tc-hydrophosphonic acid; ^(99m) Tc-monoclonal antibody tofibrin; ^(99m) Tc-monoclonal antibody to myosin; ^(99m) Tc-tissueplasminogen activator;hexakis(1-isocyano-2-methoxy-2-methylpropane)-[^(99m) Tc]technetium(I);bis[(1,2-cyclohexanedionedioximato)-(1-)-O]-[(1,2-cyclohexanedionedioximato)-(2-)-O]-methylborato-(2-)-N,N',N",N'",N"",N'""-chloro[^(99m)Tc]technetium (III); and ^(99m) Tc-glycero-glucoheptanoic acid; or apharmaceutically acceptable salt thereof.
 20. A method for radioimagingof blood pool and myocardial infarct in a subject, which comprisesadministering an effective amount of the radiopharmaceutical compositionof claim 19 by intravenous administration.
 21. The radiopharmaceuticalcomposition of claim 12 wherein the ^(99m) Tc-based radiopharmaceuticalis selected from: ^(99m) Tc-diethylenetriaminepentaacetic acid;[bis(2,3-butanedione-dioximato)(1-)-O]-[(2,3-butanedione-dioximato)(2-)-O]-(2-methylpropyl)borato-(2-)-N,N',N",N'",N"",N'""-chloro[^(99m)Tc]technetium; ^(99m) Tc-ethyl cystinate dimer; and ^(99m)Tc-3,3'-[(2,2-dimethyltrimethylene)diimino]di-2-butanone-dioxime; or apharmaceutically acceptable salt thereof.
 22. A method forbrain/cerebral perfusion radioimaging in a subject, which comprisesadministering an effective amount of the radiopharmaceutical compositionof claim 21 by intravenous administration.
 23. The radiopharmaceuticalcomposition of claim 12 wherein the ^(99m) Tc-based radiopharmaceuticalis selectedfrom:bis[N-[2-[(3-bromo-2,4,6-trimethylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)-glycyl]-[^(99m)Tc]technetium;bis[N-[2-[[2,6-bis(1-methylethyl)phenyl[amino]-2-oxoethyl]-N-(carboxymethyl)-glycyl]-[^(99m)Tc]technetium;bis[-N-(carboxymethyl)-N-[2-[2,6-dimethylphenyl)-amino]-2-oxoethyl]-glycyl]-[^(99m)Tc]technetium; and ^(99m) Tc-polyisohexylcyanoacrylate nanoparticles; ora pharmaceutically acceptable salt thereof.
 24. A method forradioimaging of the liver, gall bladder, and/or spleen in a subject,which comprises administering an effective amount of theradiopharmaceutical composition of claim 23 by intravenousadministration.